At the present time there is high demand for immunoglobulin G (IgG) which is polyvalent with a wide spectrum of human antibodies and has total functionality) neutralising capacity, opsonisation, average life conserved), with intact molecules (integrity of the crystallisable Fc fragment) and a normal distribution of IgG subclasses identical or equivalent to natural plasma, especially for the minority subclasses (IgG3 and IgG4).
The routes for the therapeutic administration of IgG may be intravenous, subcutaneous and intramuscular, and in addition to this it may be administered by other less conventional routes such as the oral, inhaled or topical routes.
Nevertheless intravenous administration offers the most useful therapeutic indications, whether for the treatment of primary immunodeficiencies or for variable common immunodeficiency (deficit of IgG and IgA subclasses) (Espanol, T. “Primary immunodeficiencies”. Pharmaceutical Policy and Law 2009; 11(4); 277-283), which is incorporated herein by reference, secondary or acquired immunodeficiencies (for example infection by viruses such as cytomegalovirus, herpes zoster, human immunodeficiency) and diseases of an autoimmune origin *thrombocytopenic purpura, Kawasaki's Syndrome, for example) (Koski, C. “Immunoglobulin use in management of inflammatory neuropathy”. Pharmaceutical Policy and Law 2009; 11(4): 307-315), which is incorporated herein by reference.
Ideally IgG for intravenous use (IGIV) should be formulated with a high concentration in liquid and preferably should be capable of being stored up to approximately 30° C. in order to facilitate conservation of the product and immediate infusion.
It has been described that in order to reduce possible IgG intolerance reactions it is necessary that immunoglobulin A (IgA) and immunoglobulin M (IgM), as well as blood group agglutinins, should be absent, or in an undetectable quantity. It is also essential that the product should be virtually free of any enzyme activity, both through the presence of plasmin or plasminogen, or prekallikrein, or its activators, kinins or kininogen, or coagulation factors such as factor XI/factor XIa, among others.
On the other hand the human origin of the starting plasma for obtaining polyvalent IgG makes it necessary to reduce the risk of infection through the transmission of viruses or pathogens to a minimum. As described by Fernandes et al. (ES 500121) and Hirao, Y. et al. (EP 196761 and EP 253313), which are incorporated herein by reference, heat treatment of IgG in solution (liquid), or pasteurisation, can be performed effectively in the presence of protectors against denaturing of the IgG (e.g., saccharose, sorbitol, amino acids). For this purpose the solution is raised to a temperature of approximately 60° C. for at least some 10 hours, activating or attenuating the most dangerous pathogens. These pathogens may have a lipid coat such as human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV), or be naked, such as poliovirus, hepatitis A virus (HAV) and parvovirus, among others (Uemura Y. et al. “Inactivation and elimination of viruses during the fractionation of an intravenous immunoglobulin preparation”. Vox Sang. 1989; 56: 155-161), which is incorporated herein by reference.
Nevertheless, pasteurisation, even in the presence of stabilisers and under the best process conditions, inevitably results in the formation of irreversible high molecular weight protein aggregates such as IgG polymers and/or polymers of other accompanying proteins, in greater or lesser proportion depending upon the purity of the starting IgG (Hirao, Y. et al., above; and Ristol, P. et al. EP 1225180 and ES 2091161), which are incorporated herein by reference.
During the decade 1960-1970 the presence of irreversible high molecular weight aggregates known as IgG polymers was associated with the consumption of complement for activation of the same (anticomplement activity, ACA) during the intravenous administration of IgG, and this phenomenon was linked to severe intolerance or anaphylaxis reactions observed (Barandum, S. et al. Vox Sang. 7: 157-174, 1962), which is incorporated herein by reference. Because of this health authorities regulated the maximum content of polymers in IGIV, or molecular forms higher than dimers, to a limit of 3% (Eur.Ph. Monograph 6.3; and CMP Core SPC for normal immunoglobulin for intravenous administration: CPMP/BPMG/859/95 rev.2), which is incorporated herein by reference. This consideration is especially important for a liquid formulation because the 3% limit must also be maintained up to the expiry date for the product. A virtually total absence of these IgG polymers must therefore be achieved, both after pasteurisation and in the final product obtained, to ensure that the product will not deteriorate over the long term and ensure the maximum possible storage temperature.
At the present time most of the liquid IgG available on the market and formulated with amino acids must maintain an acid pH to avoid aggregation (Uemura, Y. “Dissociation of aggregated IgG and denaturation of monomeric IgG by acid treatment”. Tohoku J. Exp. Med., 1983; 141: 337-349), which is incorporated herein by reference, preferably between a pH of 4.0-5.0 (Tenold, R. et al. U.S. Pat. No. 4,396,608, which is incorporated herein by reference) and at a temperature of 2-8° C. if they are stabilised with 0.2 M or 0.25 M glycine, such as those known by the trade names of Gamunex® (Grifols S A, Spain), Kiovig® or Gammagard® Liquid (both from Baxter, United States), or up to 25° C. if stabilised with 0.25 M proline, such as Privigen® (CSL Behring, Germany), in order to minimise molecular aggregation during storage (Jolles, S. et al. “Clinical uses of intravenous immunoglobulin”. Clin. Exp. Immunol. 2005 October; 142(1): 1-11; Hooper, J. A. “Intravenous immunoglobulins: evolution of commercial IVIG preparations”. Immunol Allergy Clin. North Am. 2008; 28(4): 765-778), which are incorporated herein by reference.
It has been demonstrated that an excessively acid pH over a long period of exposure favours the fragmentation of IgG, for example at a pH of 4.5 or below and at a relatively high temperature, for example at 30° C. (Vermeer, A. et al. “Thermal stability of immunoglobulin: Unfolding and aggregation of a multi-domain protein”. Biophys. J. 2000; 78: 394-404; Shukla, A. et al. “Strategies to address aggregation during protein A chromatography”. Bioprocess International, May 2005, which are incorporated herein by reference). Thus for example it has been reported in the literature that 10% IGIV compositions formulated with L-proline at a pH of 4.8±0.2 are sufficiently stable with regard to molecular aggregation, but a tendency to fragmentation with exposure time is observed. Thus at a temperature of 25° C. fragments amount on average to 3.9% after 36 months (Cramer, M. et al. “Stability over 36 months of a new liquid 10% polyclonal immunoglobulin product (IgPro10, Privigen®) stabilised with L-proline”, Vox Sang. 2009. DOI: 10.1111/j.1423-0410.2008.01143.x, which is incorporated herein by reference).
It has been described that the formulation of IgG with polyols or poly-alcohols, for example with maltose and sorbitol, prevents aggregation (Katakam, M. et al.: Aggregation of proteins and its prevention by carbohydrate excipients: Albumins and globulin. J. Pharm. Pharmacol. 1995; 47: 103-107), which is incorporated herein by reference, and because of this property IgG solutions that are stable up to 25° C. (with 10% maltose, trade name Octagam®) and up to 30° (with 5% sorbitol, trade name Flebogamma® DIF) have been formulated in a slightly acid pH range between 5.0 and 6.0 (Hirao, Y. et al., patent EP-278422), which is incorporated herein by reference.
However the presence of some sugars or derivatives in IgG formulations has been questioned in recent years (Szenczi, A. et al.: The effect of solvent environment on formation and stability of human polyclonal in solution. Biologicals, 2006; 34(1): 5-14), which is incorporated herein by reference, as some cases of serious kidney failure have been associated with the infusion of IgG preparations containing saccharose. Other disadvantages that may be presented by some immunoglobulin compositions with particular sugars (saccharose) and some high concentrations of polyols (10% maltose) is the relative capacity to increase blood viscosity when infusing the solutions, this being linked to some very serious cases of intravascular thrombosis and acute myocardial infarction where there is previous disease or the patient is at risk (Radosevich, M. and Burnouf, T. “Intravenous immunoglobulin G: Trends in production methods, quality control and quality assurance.” Vox Sang., 2009; 1-17; Katz, U. and Shoenfeld, Y.; Review: intravenous immunoglobulin therapy and thromboembolic complications. Lupus, 2005; 14(10): 802-808, which are incorporated herein by reference).
It has also been detected that some commercial IGIV products contain active procoagulating enzymes, remnants from their process of purification, which have a marked thromboembolic effect (TEE), and an association between TEE and the presence of factors XI/XIa and/or other procoagulant factors (e.g. kallikrein or the like) has been proved. Elimination of thromboembolic capacity is an imperative which must be fulfilled for IGIV infusions, with maximum guarantees of tolerance and safety.